PROJECT SUMMARY Small RNAs (smRNAs) are important non-coding regulators broadly implicated in human cancer initiation and progression. The functions and mechanism of smRNAs have been widely studied in cancer research. They are also being investigated as new therapeutic targets but the results so far are unsatisfactory, due in part to the complexity of miRNA-mRNA regulatory network coupled with cellular heterogeneity in human cancers. Thus, new tools that can co-profile smRNAs and large RNAs (lgRNAs) in large numbers of single cells can help address this long-standing challenge, add a new dimension to smRNA research, empower the discovery of new mechanisms of smRNAs to participate in cancer biology, and enable potential applications to cancer diagnostics and therapeutics. A close collaboration between PIs Fan and Lu has demonstrated, for the first time, co-sequencing of both smRNAs and lgRNAs from the same single cells (Wang et al., Nature Comm., 2019). It further showed that having paired smRNA and lgRNA profiles on the single-cell level can reveal miRNA-mediated gene regulation and new mechanisms for controlling miRNA expression and intercellular heterogeneity. However, this single-cell smRNA-lgRNA co-sequencing technology is a manual process with low throughput and high cost, limiting its potential for cancer research due to insufficient statistic power to interrogate highly heterogeneous tumor cells. In this application, we propose the advanced development of this technology to deliver a high-throughput single-cell smRNA/lgRNA co-sequencing (scSLRco-seq) technology. It employs a novel slip-transfer microdevice in combination with novel molecular barcoding scheme and downstream biochemistry for simultaneous analysis of smRNAs and lgRNAs in 1000?s of single cells. Specifically, we will (Aim 1) develop a microfluidic cross-flow patterning method to create 2,500 DNA barcode arrays for spatially-coupled capture and barcoding of small and large RNAs, (Aim2) develop a novel slip- transfer chip to integrate multi-step biochemistry workflow for high throughput scSLRCo-seq, and (Aim3) validate this technology using human and mouse myeloid leukemia models. This novel technology addresses the lack of capability for high-throughput single-cell smRNA/lgRNA co-profiling, filling a gap in single-cell omics field and enabling the study of new questions previously impossible to answer. It represents a major leap in the field and will find wide-spread use in cancer research and applications.